肌醇需求激酶1抑制剂对染矽尘大鼠肺纤维化干预作用研究  

Study on intervention effect of inositol requiring enzyme 1 inhibitor on pulmonary fibrosis of rats exposed to silica dust

作　　者：何艳玲[1] 姚三巧[1,2] 张林[3,4] 李清钊[1] 孙海霞[1] 张志宇[1] 白玉萍[1] 刘国荣[5] 郝小惠[6] 

机构地区：[1]河北联合大学公共卫生学院,河北唐山063000 [2]新乡医学院公共卫生学院,河南新乡453000 [3]山东大学附属生殖医院医务部,山东济南250000 [4]郑州大学公共卫学院,郑州450000 [5]河北联合大学附属医院,唐山063000 [6]河北联合大学医学实验中心,唐山063000

出　　处：《中国职业医学》2015年第2期147-152,共6页China Occupational Medicine

基　　金：河北省自然科学基金(H2014209114)

摘　　要：目的研究肌醇需求激酶1(IRE1)抑制剂对染矽尘大鼠肺纤维化内质网应激(ERS)凋亡途径的影响,评价IRE1抑制剂对大鼠矽肺纤维化的干预作用。方法无特定病原体(SPF)级健康成年雄性SD大鼠24只随机分为对照组、矽肺组和IRE1组,每组8只。矽肺组和IRE1组大鼠均采用非气管暴露法一次性注入质量浓度为50.0 g/L的二氧化硅混悬液1.0mL造模,对照组注入等体积灭菌生理氯化钠溶液。造模28 d后,IRE1组大鼠腹腔注射剂量为1.0 mg/kg体质量的IRE1抑制剂,其余2组予等体积灭菌生理氯化钠溶液,每7天注射1次,共4次。造模后第56天处死大鼠,收集左侧肺泡灌洗液,分离、纯化和培养肺泡巨噬细胞(AM)。观察肺组织病理变化情况,实时荧光定量聚合酶链式反应测定肺组织Ⅰ型和Ⅲ型前胶原mRNA相对表达水平,流式细胞术检测AM活性氧(ROS)阳性率和线粒体去极化率,免疫印迹法测定肺组织内IRE1α、天冬氨酸特异性半胱氨酸蛋白酶(Caspase)-12、B细胞淋巴瘤/白血病-2蛋白(Bcl-2)和Bcl-2相关X蛋白(Bax)的相对表达水平。结果肺组织病理学检查结果显示,矽肺组肺组织可见炎细胞浸润和典型矽结节形成;IRE1组可见普遍炎细胞浸润和肺泡间隔变大,有弥漫性纤维化改变,但未见矽结节形成。IRE1组、矽肺组大鼠肺组织Ⅰ型、Ⅲ型前胶原mRNA相对表达水平和AM的ROS阳性率、线粒体去极化率均高于对照组(P<0.01),IRE1组上述4个指标均低于矽肺组(P<0.01)。IRE1组、矽肺组大鼠肺组织IRE1α、Caspase-12和Bax蛋白的相对表达水平均高于对照组(P<0.01),IRE1组大鼠上述3个指标均低于矽肺组(P<0.01);但IRE1组和矽肺组大鼠肺组织Bcl-2相对表达水平均低于对照组(P<0.01),IRE1组大鼠Bcl-2相对表达水平高于矽肺组(P<0.01)。结论 IRE1参与的ERS凋亡通路可能在矽肺肺纤维化的发生和发展中发挥着重要作用;IRE1抑制剂可以通过阻断IRE1通路拮抗ERS诱导的AMObjective To study the effect of inositol requiring enzyme 1 （IRE1） inhibitor on apoptosis pathway of pulmonary fibrosis in rats exposed to silica via endoplasmie reticulum stress （ERS）, and further to evaluate the intervention effect of IRE1 inhibitor on pulmonary fibrosis in rats. Methods Totally 24 specific pathogen free healthy adult male SD rats were randomly divided into control group, silicosis group and IRE1 group, with 8 rats in each group. Rats in silicosis group and IRE1 group were given 1.0 mL of silica suspension （ mass concentration was 50. 0 g/L） to establish the silicosis model by one-time non-intratracheal instillation method, control group was treated with equal volume of sterilized physiological sodium chloride solution. Twenty-eight days after modelling, rats in IRE1 group were intraperitoneally given injection of IRE1 inhibitor at 1.0 mg/kg body weight, the other 2 groups were given equal volume of sterilized physiological sodium chloride solution, once per 7 days for 4 times. Fifty-six days after modelling, rats weresacrificed to collect the left lung bronchoalveolar lavage fluid, and the separation, purification and cultivation of alveolar macrophage （AM） were carried out. The pathological changes of lung tissue were observed, real-time fluorescence quantitative polymerase chain reaction was used to measure the mRNA relative expression levels of lung tissue of type I and type Ⅲ procollagen. Reactive oxygen species （ROS） positive rate and mitochondria depolarization rate in AM were detected by flow cytometry. Western Blotting was used to detect the relative expression levels of IRE1 alpha, Caspase-12 and B cell lymphoma./leukemia-2 protein （ Bcl-2 ） and Bcl-2 associated X protein （ Bax ）. Results Histopathologieal examination showed that the inflammatory cell infiltration and typical silicotic nodule formation were found in lung tissue of silicosis group ; IRE1 group showed widespread infiltration of inflammatory cells and alveolar septa getting larger and diffu

关 键 词：肌醇需求激酶1 抑制剂 肺泡巨噬细胞 内质网应激 矽肺 肺纤维化 

分 类 号：R114[医药卫生—卫生毒理学]